PB2476: TARGETING OF BRD4 TO COUNTERACT CYTOKINE-INDUCED EXPRESSION OF PD-L1 AND PD-L2 ON VASCULAR, OSTEOBLASTIC AND STROMAL NICHE CELLS
Authors: Yüksel Filik, Karin Bauer, Peter Valent
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance against various anti-neoplastic drugs in diverse leukemias. Recent data suggest that targeting of niche cells may be an effective approach to overcome niche-mediated resistance. However, niche cells themselves are considered to be resistant against therapeutic intervention. So far, little is known about mechanisms promoting niche related resistance against neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Aims: We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in niche cells. Methods: Flow cytometry, RT-PCR, Immunocytochemistry stainings and 3H-Thymidine Uptake Results: Expression of checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined on primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, the human stromal cell line HS5, and the macrophage cell lines Mono Mac 6 and THP1. As assessed by flow cytometry, all niche cell types were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable level in these cells. PD-L1, PD-L2 and CD47 were also found to be expressed at the mRNA level in all niche cell types examined. Of all cytokines tested (n=12), interferon-alpha (IFN-A) and interferon-gamma (IFN-G) were found to promote expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 stroma cells in our flow cytometry experiments and immunocytochemistry experiments. In addition, IFN-G enhanced surface expression of PD-L1 and PD-L2 in Mono Mac 6 and THP-1 cells. Tumor necrosis factor-alpha (TNF-A) was found to further promote IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells as determined by flow cytometry and immunocytochemistry. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 in checkpoint regulation. Finally, dBET6 suppressed the proliferation of all niche cell types tested. Summary/Conclusion: Together, we show that endothelial cells, osteoblasts, stromal cells, and macrophage cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that IFN-G and IFN-A augment expression of these immune checkpoint antigens on endothelial cells, osteoblasts and stromal cells. In addition, we show that the cytokine combinations INF-G+TNF-A and IFN-A+TNF-A further enhance expression of PD-L1 and PD-L2 in these cells. Moreover, our data show that the BRD4-targeting drugs JQ, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression. Finally, dBET6 also blocked the expansion of niche cells. Whether suppression of growth and checkpoint expression in niche cells is an effective approach to counteract niche-mediated expansion of neoplastic cells in the bone marrow and drug resistance in hematopoietic malignancies in vivo remains to be elucidated. Keywords: Bone marrow niche, Leukemia, Resistance, Cytokine